Molecular factors influencing epithelial … factors influencing epithelial-mesenchymal transition in breast cancer Gisela Nilsson Department of Medical Biochemistry and Cell Biology,

Molecular factors influencing

epithelial-mesenchymal

transition in breast cancer

Gisela Nilsson

Department of Medical Biochemistry and Cell Biology

Institute of Biomedicine

Sahlgrenska Academy at University of Gothenburg

Gothenburg 2014

Molecular factors influencing epithelial-mesenchymal transition in breast

cancer

© Gisela Nilsson 2014

[email protected]

ISBN 978-91-628-9116-9

Printed in Gothenburg, Sweden 2014

Kompendiet, Göteborg

To my family

Molecular factors influencing epithelial-mesenchymal

transition in breast cancer

Gisela Nilsson

Department of Medical Biochemistry and Cell Biology, Institute of

Biomedicine

Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden

ABSTRACT

Epithelial-mesenchymal transition (EMT) is a developmental process defined by loss

of epithelial characteristics and acquisition of mesenchymal phenotype. EMT or

similar processes are also implicated in carcinoma cell invasion and the progression

of breast carcinoma to metastasis. In a cell model system for mammary

carcinogenesis it has previously been shown that signaling from the oncogenic

receptor tyrosine kinase c-erbB2 (HER2), frequently overexpressed in mammary

cancers, induces EMT. In this system, c-erbB2-induced EMT was significantly

delayed by high cell-density and cell-cell-dissociation occurred before

downregulation of the epithelial adhesion molecule E-cadherin. Loss of E-cadherin

expression is generally viewed as a fundamental event in EMT. This thesis shows

that ectopic expression of E-cadherin concomitant with c-erbB2 signaling did not

hinder the progression of EMT. E-cadherin expressed in mesenchymal cells had a

weaker attachment to the cytoskeleton, implicating that rearrangement of the

cytoskeleton is an important mechanism in EMT-associated cell-cell-dissociation.

Expression of dominant negative E-cadherin weakened cell-cell adhesion but did not

enable EMT at high cell-density. These finding indicate that loss of E-cadherin is a

consequence rather than a cause of EMT and that density-dependent inhibition of

EMT is not mediated by E-cadherin. The expression of the transcription factor

nuclear factor I-C2 (NFI-C2) is lost during mammary tumor progression and NFI-C2

has been shown to counteract EMT by repressing the transcription factor Forkhead

box F1 (FoxF1). FoxF1 induces EMT and invasiveness in breast cancer cells. In this

thesis, Affymetrix microarray was used to find oppositely regulated targets of NFI-

C2 and FoxF1. The extracellular matrix enzyme lysyl oxidase (LOX) was found to

be negatively regulated by NFI-C2 and positively regulated by FoxF1 and

responsible for the increased invasiveness caused by FoxF1 overexpression. A

signaling pathway was identified where FoxF1-induced upregulation of LOX

activated focal adhesion kinase, subsequently suppressing Smad2 activity. In parallel,

overexpression of FoxF1 activated the p38 MAPK signaling pathway. These findings

give new insights into the regulation of signaling pathways known to be important

during breast tumor progression. Based on the findings that NFI-C2 is lost during

breast tumor progression and suppresses EMT, the prognostic value of NFI-C2 in a

mixed cohort of breast cancer patients was investigated. NFI-C2 was found to be a

powerful prognostic marker associated with good prognosis in breast cancer.

Keywords: breast cancer, epithelial-mesenchymal transition, c-erbB2, E-cadherin,

NFI-C2, FoxF1, LOX

ISBN: 978-91-628-9116-9

i

LIST OF PAPERS

This thesis is based on the following studies, referred to in the text by their

Roman numerals.

I. Nilsson GM., Akhtar N., Kannius-Janson M., Baeckström

D. Loss of E-cadherin expression is not a prerequisite for c-

erbB2-induced epithelial-mesenchymal transition.

International Journal of Oncology. 2014 Jul;45(1):82-94.

II. Nilsson G., Kannius-Janson M. Forkhead box F1 promotes

breast cancer cell migration by upregulating lysyl oxidase

and suppressing Smad2/3 signaling.

Manuscript.

III. Nilsson J., Nilsson G., Nemes S., Kovács A., Helou K.,

Jirström K., Kannius-Janson M. Nuclear factor I-C2 is a

powerful prognostic marker in breast cancer.

Manuscript.

ii

CONTENT

ABBREVIATIONS ............................................................................................. IV

INTRODUCTION ................................................................................................ 1

Mammary gland ............................................................................................ 1

Breast cancer ................................................................................................. 2

The invasion-metastasis cascade .................................................................. 3

Epithelial-mesenchymal transition ............................................................... 4

Molecular players involved in EMT ............................................................. 5

EMT in cancer .............................................................................................. 6

Important factors controlling EMT ............................................................... 7

E-cadherin ................................................................................................ 7

c-erbB2 .................................................................................................... 9

Nuclear factor I-C2 ................................................................................ 10

Forkhead box F1 .................................................................................... 12

AIMS .............................................................................................................. 14

SPECIFIC BACKGROUND ................................................................................. 15

Paper I ......................................................................................................... 15

Paper II ....................................................................................................... 16

Paper III ...................................................................................................... 17

RESULTS AND DISCUSSION ............................................................................ 18

Loss of E-cadherin expression is not a prerequisite for c-erbB2-induced

epithelial-mesenchymal transition (paper I). .............................................. 18

Forkhead Box F1 promotes breast cancer cell migration by upregulating

lysyl oxidase and suppressing Smad2/3 signaling (paper II). ..................... 20

Nuclear factor I-C2 is a powerful prognostic marker in breast cancer (paper

III). ............................................................................................................. 22

FUTURE ASPECTS ........................................................................................... 24

ACKNOWLEDGEMENTS .................................................................................. 26

REFERENCES .................................................................................................. 28

iii

iv

ABBREVIATIONS

BCSS breast cancer-specific survival

CDH-1 cadherin 1

CEL carboxyl ester lipase

CSC cancer stem cell

CTC circulating tumor cell

ECM extracellular matrix

EGF epidermal growth factor

EGFR epidermal growth factor receptor

EMT epithelial-mesenchymal transition

ER estrogen receptor

FAK focal adhesion kinase

Fox forkhead box

Jak2 janus tyrosine kinase 2

kDa kilodalton

LOX lysyl oxidase

MAPK mitogen activated protein kinase

MET mesenchymal-epithelial transition

miRNA micro-RNA

MMPs matrix metalloproteinases

NFI nuclear factor 1

NGF nerve growth factor

PDGF platelet-derived growth factor

PR progesterone receptor

RFS recurrence-free survival

RTK receptor tyrosine kinase

TGF-β transforming growth factor β

WAP whey acidic protein

Gisela Nilsson

1

INTRODUCTION

Mammary gland

The mammary gland is a secretory organ, producing milk. It consists of a

ductal network surrounded by a stroma compartment. Mammary ducts are

formed by two main types of epithelial cells; luminal and basal. The luminal

cells forms the ducts and alveoli and have secretory properties. The basal

epithelium consists of contractile myoepithelial cells that surround the

luminal epithelium. A basement membrane is lining the basal epithelium. The

stroma compartment contains extracellular matrix (ECM), stromal cells,

immune cells and adipocytes, making up the fat pad (Fig. 1) [1, 2].

Figure 1. Schematic picture of the mammary gland

The development of the human breast is a progressive process proceeding in

distinct phases; embryonic development, pre-pubertal growth, pubertal

expansion, pregnancy- and lactation-associated remodeling, and post-

lactational and post-menopausal involution [3]. The mammary gland reaches

its full mature state during the pregnancy-lactation cycle. During the distinct

phases of mammary gland development there is progressive ductal elongation

and branching followed terminal differentiation at parturition and involution

characterized by apoptosis and tissue remodeling during weaning [4, 5].

The development and function of the mammary gland are influenced by a

number of growth factors and hormones, e.g. estrogen, progesterone,

prolactin and epidermal growth factor [6, 7], and also dependent on

Molecular factors influencing epithelial-mesenchymal transition in breast cancer

2

interactions between stromal and epithelial cells. Signals from stromal cells,

as well as the composition of the ECM (laminin, fibronectin, collagen,

proteoglycans etc.) help epithelial cells to generate and maintain apico-basal

polarity [8]. In response to extracellular signals, integrins (transmembrane

receptors for ECM) activate growth and survival signals [9]. Moreover, many

of the factors important for mammary gland development are also implicated

in breast cancer. During the development of the mammary gland the

epithelium shows high levels of proliferation, reduced apico-basal polarity

and epithelial-mesenchymal plasticity, characteristics also associated with

tumorigenesis [10].

Breast cancer

Breast cancer development is a multi-step process categorized by different

stages [11]. These stages include cellular immortality, hyperplasia,

tumorigenicity and invasiveness. During these stages there is an initial

epithelial hyperplasia, which can develop into atypical hyperplasia, in situ

carcinoma, invasive carcinoma and metastasis. Breast cancer can begin in

different areas of the mammary epithelium; the ducts or the lobules and most

commonly derived from the luminal cells [12]. It is a heterogeneous disease

with different subtypes. These subtypes show differences in the molecular

profile, response to therapy and prognosis. The molecular subtypes are;

luminal A, luminal B, molecular apocrine, basal-like, HER2, normal breast-

like and claudin-low [13-15].

The mechanisms important for normal breast function are also important in

suppressing the development of tumors by regulating proliferation and

survival. Genetic and epigenetic changes leading to dysregulation of these

normal processes can cause uncontrolled proliferation and imbalance

between proliferation and apoptosis, promoting carcinogenesis. Inherited or

acquired mutations in oncogenes (e.g. HER2, MYC, CCND1, PIK3CA [16-

19]) and tumor suppressors (e.g. P53, BRCA1/2, RB1, PTEN [20-24]) are

associated with breast cancer. Hormones and growth factors controlling

normal mammary gland development together with their receptors and co-

regulators are also involved in the development of breast cancer. If these

factors are dysregulated it can lead to amplification of growth and survival

signals [25].

In addition, microRNAs (miRNAs) have been shown to play key roles in

carcinogenesis. miRNAs are small non-coding RNA molecules that control

post-transcriptional gene expression by binding to target messenger RNAs,

causing their degradation and downregulation of the target protein. Some

function as tumor suppressors and others act as oncogenes [26-28].

Gisela Nilsson

3

As a response to the genetic alterations in the epithelial cells, there are

alterations in the microenvironment, such as increased levels of growth

factors and cytokines, and increased matrix synthesis, cross-linking and ECM

stiffening. Matrix stiffening increases integrin signaling, promoting cell

growth, survival and tumor progression [29-32].

It has been suggested that different cell types or transformation of mammary

stem cells at different developmental stages are the cause of the different

breast cancer subtypes. The more aggressive breast cancer subtypes are

suggested to originate from mammary stem cells and the less aggressive

subtypes from more differentiated mammary cells [2, 33-35].

Cancer stem cells (CSCs) were first identified in hematopoietic cancers and

later in solid tumors such as breast tumors [36-38]. The existence of CSCs in

the breast was suggested in a study demonstrating that only a minority of

cancer cells have the ability to form new tumors. Tumorigenic (cancer-

initiating) and non-tumorigenic cancer cells were identified based on

differences in molecular markers [38]. Tumorigenic cancer cells have the

ability to self-renew and produce progeny that are able to differentiate, which

are properties of normal stem cells [39]. CSCs also have metastatic properties

such as increased motility [40].

The invasion-metastasis cascade

Metastases are formed by a multi-step process. Cancer cells that have

acquired the ability to disseminate from the primary tumor can travel through

the blood and lymphatic system to distant sites in the body where they can

form new colonies (metastases). The process when carcinoma cells acquire

the ability to break through the basement membrane and invade the nearby

stroma is termed localized invasion. The step when cancer cells enter the

blood and lymphatic vessels is called intravasation. The migrating cells may

die from anoikis, a form of apoptosis triggered by detachment from the ECM.

They can also survive for long periods and in some instances extravasate into

the surrounding tissue, leading to formation of micrometastases. If the

foreign tissue microenvironment is favorable, the cancer cells may begin to

proliferate and form a secondary tumor, a process termed colonization (Fig.

2) [41]. Carcinomas are benign as long as the tumor cells do not break

through the basement membrane and malign when they have acquired the

ability to metastasize which is the leading cause of death in cancer patients.


4

Figure 2. Schematic picture of cancer cell invasion and metastasis. 1. Normal

epithelium. 2. Carcinoma in situ, 3. Invasive carcinoma. 4. Intravasation. 5.

Extravasation. 6. Micrometastasis. 7. Colonization.

Epithelial-mesenchymal transition

Epithelial and mesenchymal cell phenotypes are not always permanent.

During development it is critical that cells can change in morphology and

behavior for proper morphogenesis. They do this by converting between the

epithelial and mesenchymal phenotypes. These processes are termed

epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial

transition (MET) [42]. EMT and MET are critical during developmental

stages such as gastrulation [43], neural crest formation [44], and heart valve

formation [45], as well as other morphogenetic events. EMT has also been

observed during branching morphogenesis in the mammary gland [46].

EMT involves activation of genes that are critical for creating the

mechanisms needed for cell migration through the ECM. Epithelial cells are

attached to each other by different junctions (tight junctions, adherens

junctions and desmosomes). During EMT, these junctions dissolve, allowing

epithelial cells to separate, lose apical- basal polarity and gain motility. The

epithelial cell-cell adhesion molecule E-cadherin, and epithelial-specific

integrins, are replaced by mesenchymal specific N-cadherin and integrins

specific for a more transient adhesion. The epithelial intermediate filaments,

cytokeratins, are replaced by vimentin [47]. The underlying basement

membrane is degraded by matrix metalloproteinases (MMPs) and the cell

migrates into the surrounding stroma (Fig. 3). After migration to a distant

site, the mesenchymal cells may return to a epithelial phenotype by

undergoing MET [48].

Gisela Nilsson

5

Figure 3. Epithelial-mesenchymal transition. To migrate and to invade a cell needs

to detach from the neighboring cells in the epithelium. E-cadherin and epithelial

adhesion complexes are replaced by mesenchymal adhesion complexes. Epithelial

cells lose their polarity and gain motility and a mesenchymal morphology. The

cytoskeleton is remodeled and vimentin is expressed. There is an increase in

expression and secretion of extracellular components, e.g. fibronectin. The figure is

adapted from [49].

Besides being critical during development, EMT is involved during wound

healing. Keratinocytes at the edge of a wound undergo EMT and MET during

the wound healing process [50]. EMT or EMT-like events have also been

implicated in fibrosis [51] and cancer metastasis [52]. There are similarities,

but also differences, between EMT events during development and other

processes, leading to the classification of three different EMT types. Type 1

refers to developmental EMTs; type 2 includes EMT events in wound healing

and fibrosis; and type 3 refers to the EMT associated with cancer metastasis.

One difference between type 2 and 3 to type 1 is the existence of an

inflammatory response, which does not exist in embryos [53].

Molecular players involved in EMT

EMT is driven by a network of transcription factors including Snail1, Snail2

(also known as Slug), and Snail3, Zeb1 and Zeb2, Twist1 and Twist2 and

others. Their expression is triggered in response to extracellular signals

derived from the cellular microenvironment. Potent inducers of EMT are

members of the transforming growth factor–β (TGF-β) family [54, 55],

epidermal growth factor (EGF) [56, 57], fibroblast growth factor (FGF) [58,

59] and platelet-derived growth factor (PDGF), but also Wnt [60], Notch

[61], Hedgehog [62] and nuclear factor-κB (NF-κB) [63]. Receptor tyrosin

kinases (RTKs), as well as other types of receptors, and a wide range of

signaling pathways transduce EMT initiating signals and several pathways

work in cooperation in order to full transition to a mesenchymal phenotype.

Activation of Ras and Src pathways are involved in dissociation of adherens


6

junctions and desmosomes and remodeling of cytoskeleton by regulating the

Rho family of GTPases [64]. Hypoxia (oxygen deprivation in the

microenvironment) can also drive EMT during development or tumor

progression. The hypoxic response is mainly mediated by hypoxia inducible

factor-1 (HIF-1) which can promote EMT by regulation of Twist [65].

miRNAs have also emerged as key players in EMT and MET. The miRNA-

200 family are proposed to be involved in reversible switching between

epithelial and mesenchymal state [66].

EMT in cancer

In cancer, genetic and epigenetic events lead to alterations in cancer cell

phenotype. These changes become most evident when cancer cells leave the

epithelium and initiate metastasis (Fig. 2 and 3). Carcinoma cells at the

invasive front of a tumor may lose epithelial traits and acquire a

mesenchymal phenotype. Signals supplied by stromal cells, that secrete

growth factors and cytokines, also contributes in the transition to

mesenchymal phenotype [30, 67].

Signaling pathways and downstream effectors involved in the phenotypic

changes needed for a carcinoma cell to detach from the epithelium seems to

be highly similar to the events that orchestrate developmental EMT.

Therefore, EMT or EMT-like events have been implicated in tumor invasion

and metastasis, especially in the early steps of metastasis by facilitating cell

dissemination and thereby generation of circulating tumor cells (CTCs). The

revers process, MET, is believed to be responsible for the formation of

metastases at distant organs [68]. Constitutive expression of transcription

factors associated with EMT, suppresses metastasis formation, suggesting

that EMT transcription factors must be downregulated for metastases to form

[69-71]. In addition, high expression of E-cadherin is seen in breast cancer

metastases [72, 73], suggesting that re-expression of epithelial markers is

necessary for formation of metastases.

It is difficult to study EMT in human tumors, since EMT is considered a

transient event and a tumor specimen represents only one instant. However,

EMT in cultured cells and animal models has been intensely studied.

Multiple signaling pathways and often cooperation of different pathways has

been shown to be involved in the initiation and progression of EMT in cancer

cell models. Overexpression of RTKs, e.g. EGF receptors, or constitutive

signaling from Ras, can drive EMT by activating Raf/MAPK- and PI3K

pathways leading to expression of transcription factors like Snail, Slug Twist

and Zeb1 [74-76]. There are many studies implicating TGF-β as a master

Gisela Nilsson

7

regulator of EMT, and cooperation of TGF-β with Ras or RTKs has been

shown to cause EMT [77].

Expression of mesenchymal markers have been observed in breast

carcinomas of the basal and triple-negative subtypes [78]. However, such

tumors also contain epithelial markers, suggesting that they have undergone a

partial EMT [79, 80].

It has been proposed that EMT can generate CSCs [81], however the origin

of CSCs is a subject of debate. It has been shown that in mammary epithelial

cells, expression of Twist1 or Snail, or treatment with TGF-β increases the

number of cells with CSCs properties (CD44high

/CD24low

) [82, 83]. EMT

markers and stem cell markers are coexpressed in CTCs from breast cancer

patients with metastasis [84, 85] .

Important factors controlling EMT

As described above there are many factors involved in EMT. The studies

included in this thesis have focused specifically on four different factors that

have been shown to be involved in breast tumor development and EMT. The

following sections in the introduction will be about these factors.

E-cadherin

E-cadherin (E for epithelia), also known as cadherin-1 (CDH-1), uvomorulin

and L-CAM, belongs to the type-1 classical cadherin family and is the major

cell adhesion molecule (CAM) involved in binding between neighboring

cells in adherens junctions.

Cadherins play a central role during the development and maintenance of

multicellular organisms. E-cadherin helps to provide mechanically strong

adhesive links between cells in the tissue and is important in defining apico–

basal polarity and maintaining epithelial morphology. E-cadherin is

expressed early in embryonic development and initiates polarization of cells

to form the blastocyst [86]. E-cadherin knockout studies in mice show

lethality at the blastocyst stage [87]. Conditional gene inactivation of E-

cadherin in the mouse mammary gland leads to altered epithelial

differentiation and cell death [88].

The extracellular domain of E-cadherin consists of five repetitive domains.

The outermost part of E-cadherin contains an adhesion recognition motif

important for binding specificity to identical cadherin molecules [89]. E-

cadherin molecules form Ca2+

dependent complexes, also involving catenins;

β-catenin/α-catenin or γ-catenin (plakoglobin)/α-catenin and p120 catenin,

mediating linkage to the actin cytoskeleton (Fig. 4) [90-92]. β-catenin and γ-

catenin both interact with E-cadherin but since their binding sites overlap


8

they cannot bind simultaneously. α-catenin is an actin binding protein and is

believed to link the catenin-cadherin complex to the cytoskeleton [93].

However, it has been demonstrated that α-catenin cannot bind actin and β-

catenin at the same time [94, 95], suggesting additional complexity.

Figure 4. Schematic drawing of the organization of E-cadherin in adherens

junctions. E-cadherin is a 120 kDa protein expressed on the surface of epithelial

cells. E-cadherin contains five extracellular domains, a single transmembrane

domain and a cytoplasmic domain which interacts with catenins connecting E-

cadherin to the cytoskeleton; β-catenin (β), γ-catenin (γ) and α-catenin (α). p120

catenin binds to juxtamembrane domain of E-cadherin. E-cadherins exerts

homophilic interactions between neighboring cells, forming a “zipper” structure.

Regulation of adherens junction formation and stability involves both

transcriptional and posttranscriptional regulation of E-cadherin. The levels of

E-cadherin expression [96] and the levels of cytosolic β-catenin regulate the

strength of adhesion [97]. The amount of E-cadherin available for adhesion

can also be regulated by recycling of endocytosed E-cadherin [98] and

proteolytic cleavage [99].

E-cadherin expression is lost during the development of most epithelial

cancers. Loss of E-cadherin expression is suggested to be one of the key steps

in EMT, invasion and metastasis [100]. Consequently, E-cadherin is referred

to as a tumor suppressor and forced re-expression of E-cadherin in carcinoma

cells can reverse malignant properties [101]. Loss of E-cadherin expression is

a typical feature of invasive lobular carcinoma [102]. However, other types of

breast cancer show variable downregulation of E-cadherin expression [103,

104].

Gisela Nilsson

9

c-erbB2

The c-erbB family of RTKs, includes epidermal growth factor receptor

(EGFR; c-erbB1, HER1), c-erbB2 (Neu, HER2), c-erbB3 (HER3) and c-

erbB4 (HER4), which are activated by a number of ligands, e.g. EGF, TGF-α

and heregulin. c-erbB receptors consist of an extracellular ligand binding

domain, a single transmembrane domain and an intracellular kinase and

substrate domain. Upon ligand binding they form a variety of homo- or

heterodimers leading to activation and phosphorylation [105]. Activation of

these receptors induces cell growth, proliferation, differentiation and survival

signals, including Rho GTPases, the Ras-MAP kinase cascade, PI3K and

phospholipase Cγ [106]. c-erbB2 and c-erbB3 activity require

heterodimerization with other c-erbBs since c-erbB2 does not bind growth

factor ligands, and c-erbB3 lacks intrinsic kinase activity [107]. The c-erbB

family plays crucial roles in heart and nervous system development [108-

111] and epithelial morphogenesis, for example in the development of

embryonic and adult mammary gland [112-114].

c-erbB2 is a glycosylated protein with a molecular weight of 185 kDa.

Amplification of c-erbB2 is a common feature in breast cancer.

Overexpression is seen in 15-30 % of breast tumors and is associated with

aggressive and invasive tumors and poor prognosis [16, 115]. Treatment with

the monoclonal antibody trastuzumab (Herceptin) against c-erbB2 has a

positive effect on overall survival and recurrence risk [116, 117]. Lapatinib, a

tyrosine kinase inhibitor, is also used in treatment of c-erbB2 positive breast

cancer [118]. Overexpression of the normal c-erbB2 protein, or point

mutations in its transmembrane domain, has been shown to have a

transforming effect on mammary epithelial cells. When overexpressed, c-

erbB2 can form active homodimers in the absence of ligand [119-121].

Association between c-erbB2 homodimerization and gene amplification has

been shown in vivo, which also was associated with poor prognosis [122]. c-

erbB2 affects cell adhesion and migration, implicating a role for c-erbB2 in

metastasis [123, 124]. Although the receptor commonly associated with EMT

is the TGF-β receptor, there are studies also implicating c-erB2 in EMT. In

an in vitro three-dimensional model system it was demonstrated that

overexpressed c-erbB2 induces proliferation and disrupts tight junctions and

epithelial polarity [125]. Overexpression of c-erbB2 in the mammary

epithelial cell line MTSV1-7 inhibited transcription of E-cadherin [126]. In

vivo selection of MCF-7 cells overexpressing c-erbB2 in nude mice, resulted

in a sub-line that had underwent EMT [127].

CTCs expressing EMT- and stem cell-associated markers have been detected

in the blood of patients with c-erbB2 positive metastatic breast cancer [128].

As mentioned, EMT has been proposed to generate CSCs [81]. CSCs often


10

exhibit resistance to therapy [129]. It has been suggested that the mechanisms

leading to resistance to the c-erbB2-targeting drugs trastuzumab and lapatinib

in c-erbB2 positive breast cancer may be linked to induction of EMT [130,

131].

In paper I, we have investigated the role of E-cadherin in c-erbB2-induced

EMT.

Nuclear factor I-C2

Nuclear factor I-C2 (NFI-C2) is a member of the Nuclear factor I (NFI)

transcription factor family. The NFI gene family consists of four members,

NFI-A, NFI-B, NFI-C and NFI-X. NFI gene transcripts are differentially

spliced [132] and expressed in unique but overlapping patterns [133-135].

The NFI proteins bind DNA as dimers at the consensus sequence

TTGGC(N5)GCCAA and function as transcriptional activators as well as

repressors of target genes [136]. NFI proteins are involved in regulation of

genes controlled by e.g. insulin [137], TGF-β [138, 139], cAMP [137],

steroid hormones [140], TNFα [141] and others. The unique expression

patterns of NFI proteins suggests that they may play distinct roles in

regulating tissue-specific gene expression during mammalian embryogenesis

[134] and they have been demonstrated to be involved in cell differentiation

[142, 143]. The oncogene Ras causes transformation of cells. It has been

demonstrated that NFI can suppress Ras-induced transformation.

Overexpression of NFI genes in chicken embryo inhibited transformation by

subsequent overexpression of Ras in these cells [144]. Conversely,

overexpression of Ras in mouse cells represses expression of NFI by

affecting mRNA stability [145]. These studies suggest that loss of NFI

function may promote tumor development. Furthermore, NFI proteins have

been implicated in the function of the mammary gland after the identification

of NFI binding sits in the genes for the milk proteins whey acidic protein

(WAP) and carboxyl ester lipase (CEL) [146, 147].

Identification of specific NFI family members and isoforms involved in

different processes has been hindered by the lack of specific antibodies. The

responsible specific NFI protein is therefore unidentified in most studies.

Our laboratory has demonstrated that the specific isoform NFI-C2 is the

protein responsible for activation of the WAP and CEL promoters in the

mouse mammary gland at mid-pregnancy [148]. Further studies suggest a

broader role for NFI-C2 in the mouse mammary gland. Besides activation of

milk genes, it was shown that NFI-C2 activates the tumor suppressor gene

p53 at mid-pregnancy [149]. From these studies it was suggested that NFI-C2

might participate both in the establishment of a functional gland and in the

protection of the gland against tumorigenesis during proliferation.

Gisela Nilsson

11

The polypeptide hormone prolactin plays a critical role both in the

differentiation of the gland during pregnancy and in the regulation of milk

protein gene expression [150]. The prolactin receptor can activate a number

of signaling pathways [151]. In mammary epithelial cells, prolactin binding

to the prolactin receptor activates Jak2 (Janus tyrosine kinase 2). Activated

Jak2 phosphorylates Stat5 (signal transducer and activator of transcription 5)

that dimerizes and translocates to the nucleus where it binds and activates

target genes [152]. The prolactin signaling pathway has also been

demonstrated to play a role in maintenance of the NFI-C2 protein in

mammary epithelial nuclei by regulating NFI-C2 protein levels. This occurs

by a mechanism distinct from prolactin’s regulation of Stat5 [153]. Further

studies demonstrated that prolactin via nuclear Jak2 regulates the amount of

active NF1-C2 through tyrosine phosphorylation and protection against

proteasomal degradation (Fig. 5) [154].

Figure 5. Proposed model of the prolactin-nuclear Jak2/NFI-C2 signaling pathway in

mammary epithelial cells. Activation of Jak2 by prolactin/prolactin receptor (Prolactin-R)

translocates Jak2 to the nucleus by an unknown mechanism. In the nucleus, Jak2

phosphorylates NF1-C2 on tyrosine(s) and protects it from proteasomal degradation. The

classical way by which prolactin transduces its signal in mammary gland is through the

Jak2-Stat5 pathway. The figure is reprinted with permission from Nilsson et al [154].

The prolactin-nuclear Jak2/NFI-C2 signaling pathway was later implicated in

the regulation of tumor development and EMT. It was shown that patients


12

with nuclear NFI-C2 in their breast cancer cells have better prognosis

compared to those without detectable NFI-C2, and that NFI-C2 suppresses

invasiveness and EMT by repressing the transcription factor Forkhead box F1

(FoxF1) [155]. Paper II and paper III are continuations of that study, which

will be discussed in more detail below.

Forkhead box F1

Forkhead box (Fox) proteins constitute a family of transcription factors that

are related through the presence of a conserved forkhead or winged-helix

DNA-binding domain. Fifty Fox genes have been identified in the human

genome (FoxA to FoxS). Fox proteins bind DNA at the recognition core

motif 5’ ((G/A)(T/C)(C/A)AA(C/T)A) 3’. Nucleotides outside this core motif

are important for the specificity between the different Fox proteins [156].

Temporal and spatial restriction of expression patterns provides an additional

level of specificity, as well as context dependence. For example, in the

developing lung, FoxF1 and FoxF2 have overlapping but not identical

expression patterns, suggesting that these transcription factors may have both

similar and independent functions [157, 158].

FoxF1 acts as a transcriptional activator or repressor of target genes [157,

159]. Early in development FoxF1 is required for differentiation of extra-

embryonic and lateral plate mesoderm and for mesodermal proliferation in

the primitive streak. Inactivation of FoxF1 in mouse embryos is lethal and the

embryos have abnormalities in the coelom, the amnion fails to expand and

there is a lack of vasculogenesis [160]. Later in development, FoxF1 is

expressed in the mesenchyme adjacent to the epithelium in the alimentary,

respiratory and urinary tracts, where it is believed to regulate mesenchymal-

epithelial interactions [157]. In the developing lung, FoxF1 transcription is

activated by Sonic hedgehog signaling [161, 162]. The effects of FoxF1 have

been shown to be dependent on gene dosage [163]. FoxF1 heterozygous

mouse pups show developmental defects in the gut, lungs, trachea, esophagus

and gallbladder [161, 163-165]. In the adult, expression of FoxF1 is found in

a range of tissues, e.g. in the gut, brain, eye and lung [166, 167]. In humans,

the neonatal lethal disorder Alveolar capillary dysplasia with misalignment of

pulmonary veins (ACDMPV) is linked to inactivating mutation of FoxF1

[168].

Fox proteins are important in a variety of biological processes, including

metabolism, development, differentiation, proliferation, apoptosis and

migration. As a consequence, loss or gain of Fox function can lead to

tumorigenesis. Fox proteins have been suggested to act as either tumor

suppressors or oncogenes [169].

Gisela Nilsson

13

The first study demonstrating a role of FoxF1 in tumorigenesis came from

our laboratory. There, it was shown that in a mouse mammary epithelial cell

line, FoxF1 overexpression induced EMT, migration and invasiveness. In

addition, it was demonstrated that forced expression of FoxF1 enhanced

xenograft tumorigenesis in nude mice [155]. FoxF1 has been shown to be

important for mesenchymal cell migration by directly repressing integrin β3

expression [159]. FoxF1 has also been shown to play a role in lung cancer

associated fibroblasts, where FoxF1 expression stimulated cell migration and

xenografted tumor growth [170]. In paper II, we have investigated the

mechanisms responsible for FoxF1-enhanced invasion.


14

AIMS

The objective of this thesis was to study the influence of different molecular

factors on the EMT process associated with breast cancer cell invasion and

metastasis and their possible use as biomarkers.

The aim of paper I was to study the influence of oncogenic tyrosine kinase

receptor signaling on EMT; specifically, to investigate the role of E-cadherin

during c-erbB2-induced EMT, and in high cell-density dependent inhibition

of EMT.

In paper II, the objective was to study the role of NFI-C2 and FoxF1 in

mammary carcinogenesis by identifying factors regulated by these

transcription factors and involved in EMT and invasion and elucidate what

mechanisms are responsible for FoxF1-enhanced invasion.

Paper III aimed to investigate the prognostic value of NFI-C2 in breast

cancer.

Gisela Nilsson

15

SPECIFIC BACKGROUND

Paper I

The human mammary epithelial cell line HB2 [171] is a subclone from the

non-tumorigenic MTSV1-7 cell line. MTSV1-7 cells were obtained from

luminal epithelial cells from milk and immortalized with the SV40 T antigen

[172]. HB2 cells form spherical colonies in collagen and has been used for in

vitro studies of branching morphogenesis [171]. Constitutive overexpression

of c-erbB2 in MTSV1-7 cells causes irreversible changes in morphology and

repression of E-cadherin [126, 173]. To study the order of events in c-erbB2-

signlaing effects, an inducible system in MTSV1-7 cells and HB2 cells was

developed by Baeckström et al [174], using the hybrid receptor construct trk-

neu [175]. Trk-neu consists of the extracellular domain of the trkA nerve

growth factor (NGF) receptor and the transmembrane and cytoplasmic

domain of c-erbB2 (neu). c-erbB2 homodimerization and activation can be

induced by NGF treatment of the cells (Fig. 6).

Figure 6. The trk-neu hybrid receptor. The extracellular domain of trkA nerve

growth factor (NGF) is fused to the transmembrane and cytoplasmic domains of c-

erbB2 (neu). NGF treatment induces homodimerization and tyrosine kinase (TK)

activation and mimics c-erbB2 homodimerization.

Induced c-erbB2 signaling inactivated integrin α2β1 leading to scattered

growth in collagen. In addition, prolonged activation of c-erbB2 led to EMT

[174]. Further studies using the high expressing clone HB2/tnz34 showed

that c-erbB2-induced integrin inactivation was mediated by MEK/ERK

signaling and PI3K dependent ILK and PKB signaling pathways [176].

Subsequent studies demonstrated that the EMT caused by prolonged

induction of c-erbB2 signaling in HB2/tnz34 cells was irreversible and


16

associated with anchorage independent growth [177, 178]. Furthermore, c-

erbB2-induced EMT was suppressed when cells were grown at high density.

It was hypothesized that E-cadherin homophilic interaction can suppress

EMT. It was also observed that the initial cell-scattering associated with

EMT occurred before downregulation of E-cadherin [177].

In paper I, we aimed to analyze a possible role for E-cadherin in cell-cell

contact-inhibition of c-erbB2-induced EMT.

Paper II

In paper II we used three different cell lines; HC11, MDA-MB-436 and HB2.

HC11 cells are a mouse epithelial cell line cloned from the COMMA-1D cell

line, which is derived from mammary tissue of BALB/c mice at mid-

pregnancy [179, 180]. HC11 cells have been used for in vitro studies of

epithelial cell proliferation, signal transduction and differentiation. MDA-

MB-436 is a mesenchymal human breast carcinoma cell line derived from

metastatic site (pleural effusion) and have been used for studying triple-

negative breast cancer [181]. HB2 cells are described in the previous section.

As mentioned, in a previous paper it was demonstrated that the

prolactin/nuclear Jak2/NFI-C2-pathway plays a role in breast cancer by

regulating EMT, motility and invasion [154]. In order to study the effects of

NFI-C2 without the involvement of other effectors stimulated by prolactin, a

stable form of NFI-C2 (NFI-C2S) was stably transfected in HC11 cells. A

point mutation protects NFI-C2S from proteasomal degradation in the

absence of nuclear Jak2. NFI-C2S expression in HC11 cells increased

epithelial characteristics and impeded migration. NFI-C2S expression in

mesenchymal MDA-MB-436 cells lead to loss of some mesenchymal

characteristics, decreased in vitro invasion and abolished xenografted tumor

growth in nude mice, suggesting that NFI-C2 is a suppressor of tumor

progression and EMT. This was underscored by the observation that NFI-C2

status of primary breast cancer correlated with survival and its expression

was lost in lymph node metastases (discussed in the next section, paper III).

Affymetrix microarray analysis of the transcriptome of MDA-MB-436 cells

expressing NFI-C2S or control vector identified the FoxF1 gene to be one of

the most downregulated by NFI-C2, and it was also shown that FoxF1 is a

direct target of NFI-C2. FoxF1 overexpression in HC11 cells induced EMT,

increased in vitro invasion capacity and enhanced xenografted tumorigenesis

in nude mice.

In paper II, we wanted to further study the roles NFI-C2 and FoxF1 in EMT

and invasion, primarily by identifying targets that are oppositely regulated by

these factors and involved in EMT and invasion.

Gisela Nilsson

17

Paper III

Prognostic markers are used in the evaluation of a cancer patient’s risk of

relapse and disease progression. Prognostic markers include tumor size,

axillary lymph node status, histologic grade, patient age and menopausal

status. In addition, molecular markers are used to further help to predict

prognosis or treatment response, e.g. Ki67, estrogen receptor (ER),

progesterone receptor (PR) and HER2. However, these factors are unable to

accurate predict which tumor will recur and to indicate responses to different

therapies and therefore there is a need to find new prognostic markers.

It has previously been demonstrated that the presence of NFI-C2 in breast

tumors has a clinical relevance [155]. The status of NFI-C2 was analyzed by

immunohistochemistry in a tissue microarray containing 292 samples from

patients diagnosed with stage II invasive breast cancer. Tumor cells had a

weaker staining of NFI-C2 compared to normal glandular cells and only 1 of

159 lymph node metastases stained positive for NFI-C2. Patients with NFI-

C2 in their tumor cells (74 of 292) had better prognosis compared to those

without detectable NFI-C2, suggesting that NFI-C2 is a tumor suppressor.

Furthermore, there was a correlation of nuclear Jak2 and NFI-C2 in tumor

biopsies, indicating that the regulation of the nuclear levels of NFI-C2 by the

prolactin/Jak2/NFI-C2 pathway also occurs in cancer.

These findings, together with the finding that NFI-C2 suppresses EMT, make

NFI-C2 a candidate as a prognostic marker. In paper III, we aimed to

investigate the prognostic value of NFI-C2 in a mixed cohort of breast cancer

patients. We also analyzed the levels of NFI-C2 protein after tamoxifen

treatment in cellular extracts from T47D and 4T1 cells. T47D is a human

breast cancer cell line derived from metastatic site (pleural effusion). This

cell line has epithelial morphology and is non-invasive. 4T1 is a mouse

mammary tumor cell line with epithelial morphology. 4T1 cells form highly

metastatic tumors when injected to BALB/c mice and are frequently used as a

model for metastatic breast cancer. This is also a luciferase based model and

the metastatic process can be followed by using bioluminescence in live

animals.


18

RESULTS AND DISCUSSION

Loss of E-cadherin expression is not a prerequisite

for c-erbB2-induced epithelial-mesenchymal

transition (paper I).

In paper I, we have further investigated earlier observations that cells

undergoing the first scattering phase in c-erbB2 induced EMT still expressed

surface-bound E-cadherin, and that the progression of EMT was delayed by

cell-cell contact [177]. These observations raised the questions whether loss

of E-cadherin expression is a cause or a consequence of EMT and if cell

density-dependent suppression of EMT is mediated by E-cadherin binding

between cells.

In order to analyze a possible modulation of c-erbB2-induced EMT by E-

cadherin, an inducible system for ectopic expression of E-cadherin in cells

stably expressing the trk/neu hybrid receptor (HB2/tnz34) was created. This

generated the clones TrE-ep1 and TrE-ep5 (ep for epithelial morphology).

TrE-ep5 cells were grown at low density with a combination of c-erbB2

signaling and ectopic E-cadherin expression. By subsequently analyzing

changes in expression and localization of epithelial- and mesenchymal

markers, using flow cytometry and immunofluorescence, we could show that

the forced expression of E-cadherin did not prevent the progression of EMT,

as compared to cells with c-erbB2 signaling only. EMT requires loss of cell-

cell adhesion and since E-cadherin is a major component in cell-cell contacts,

loss of its expression is considered to be a fundamental event. However, it

has been observed also in other model systems that EMT can occur without

simultaneous downregulation of E-cadherin. For example, EGF-induced

EMT in MDA-MB-468 cells occurred without complete downregulation of

E-cadherin [182, 183]. In the present study, we showed that cell scattering

following overnight EGF treatment of MDA-MB-468 cells occurred without

major changes in surface-bound E-cadherin, implicating that downregulation

of E-cadherin is not essential in at least the early phase of EMT.

In a fibroblastic clone (TrE-fib), isolated after prolonged c-erbB2 signaling in

the presence of E-cadherin, the endogenous CDH1 gene had been silenced.

Induced expression of ectopic E-cadherin did not have any effect on

morphology in these cells, suggesting that E-cadherin had lost its function.

Performing dissociation assays on confluent TrE-ep5 and TrE-fib cells

showed that induced expression of E-cadherin increased cell-cell adhesion in

TrE-ep5 cells. In contrast, in fibroblastic TrE-fib cells, E-cadherin did not

Gisela Nilsson

19

have a restoring effect on cell-cell adhesion. These results further supported

our assumption that the function of E-cadherin is lost in fibroblastic cells. In

addition, the attachment of E-cadherin to the cytoskeleton was weaker in

fibroblastic cells and β-catenin and α-catenin were localized to the cytoplasm

and nucleus, although the localization of E-cadherin was grossly the same in

epithelial and fibroblastic cells. It has previously been shown that c-erbB2

signaling in HB2 cells results in destabilization of the cortical cytoskeleton

[184]. Rearrangements of the cytoskeleton may lead to difficulties in

supporting E-cadherin mediated cell-cell adhesion, allowing cells to separate

even in the presence of surface-bound E-cadherin.

The observation that the progression of c-erbB2-induced EMT was delayed

by cell-cell contact raised the question whether cell-cell contact results in

intracellular signaling events that hinder the EMT process. If such cell-cell

contact-dependent signaling events are mediated by E-cadherin, then

interfering with E-cadherin function would allow cells grown at high density

to undergo c-erbB2-induced EMT. To test this hypothesis, we expressed a

mutant E-cadherin, where the adhesive function is abolished [90]. Cells

expressing this mutant construct, TrEwv

-ep, showed a decrease in cell-cell

adhesion. However, the combination of c-erbB2 signaling and mutant E-

cadherin expression in high density cultures did not result in progression of

EMT. This indicates that E-cadherin is not involved in mediation of density-

dependent inhibition of c-erbB2-induced EMT.

It has been demonstrated that MMP3 and Rac1b, factors that stimulate

alterations in the cytoskeleton, induce EMT in mammary epithelial cells. This

MMP3/Rac1b-induced EMT was dependent on the cells ability to spread

over a larger surface. When the available surface area was limited

MMP3/Rac1b-induced EMT was blocked [185], suggesting cell shape as an

important factor in the initiation of EMT.

Our findings suggest that loss of E-cadherin expression may be a

consequence rather than a cause of c-erbB2-induced EMT, and further that

density-dependent inhibition of c-erbB2-induced EMT is not mediated by E-

cadherin. We propose that cytoskeletal rearrangements in the initial phase of

EMT may be the mechanism leading to cell-cell separation by impairment of

E-cadherin function and cell-cell adhesion. These rearrangements might also

be important in density-dependent inhibition of EMT by eliciting signals, in

crowded cells, that control EMT progression.


20

Forkhead Box F1 promotes breast cancer cell

migration by upregulating lysyl oxidase and

suppressing Smad2/3 signaling (paper II).

The aim of this study was to further investigate the roles of NFI-C2 and

FoxF1 in EMT and invasion. Using Affymetrix microarray analysis, we

could compare the transcriptome of HC11 wild type cells to that of cells

overexpressing NFI-C2S or FoxF1 and find targets that are oppositely

regulated by these factors, involved in EMT and invasion. In order to identify

factors that might be generally important in EMT and regulated by NFI-C2,

we used the data from a previous array where the transcriptome of MDA-

MB-436 cells was compared to that of MDA-MB-436 cells overexpressing

NFI-C2S [155]. 45 genes were downregulated 1.5 fold or more by NFI-C2 in

both these cell lines. Of these 45 genes, 17 were upregulated 1.5 fold or more

by FoxF1 overexpression in HC11 cells. The gene that was by far most

upregulated by FoxF1 was lysyl oxidase (LOX). LOX is an ECM enzyme that

catalyzes the cross-linking of collagens or elastin, thereby controlling the

structure and tensile strength of the ECM, but is also implicated in EMT and

tumor progression to metastasis [186-189]. LOX has been demonstrated to be

upregulated in invasive breast cancer cell lines and breast carcinomas [190,

191]. LOX was therefore a potential factor involved in the enhanced in vitro

invasion capacity observed following FoxF1 overexpression in HC11 cells.

We could detect high levels of secreted LOX in culture media from

HC11FoxF1 cells. In addition, we could show that the enhanced in vitro

invasion capacity of HC11FoxF1 cells was mediated by LOX, as treatment

with a LOX inhibitor or reduction of LOX expression using RNAi,

significantly decreased the capacity of HC11FoxF1 cells to migrate through

matrigel. Taken together, we found LOX to be downregulated by NFI-C2 and

upregulated by FoxF1 and responsible for the increased invasion capacity of

HC11FoxF1 cells. These findings together with the previous observations

that FoxF1 is repressed by NFI-C2 which expression is lost during tumor

progression, suggests that loss of NFI-C2 may lead to expression of factors

like FoxF1 and LOX leading to EMT and carcinoma cell dissemination.

Several of the 17 genes that were shown to be downregulated by NFI-C2 and

upregulated by FoxF1 are known to be involved in TGF-β signaling and

TGF-β associated EMT. In addition, LOX has been shown to be involved in

TGF-β induced EMT [192]. This raised the question whether FoxF1 is

involved in the regulation of TGF-β signaling pathways.

TGF-β is important for mammary gland development and function and in the

suppression of tumorigenesis. Breast cancer progression can convert the

function of TGF-β to become a tumor promoting factor [193, 194]. The

Gisela Nilsson

21

oncogenic activity of TGF-β is suggested to be a result from imbalance

between canonical (Smad2/3) and non-canonical (non-Smad) TGF-β

signaling systems, where the non-canonical pathways are believed to promote

EMT and invasive and metastatic properties of cancer cells. The non-Smad

pathways include various branches of MAP kinase pathways, e.g. p38 MAPK

[195].

We investigated whether TGF-β signaling pathways are affected by FoxF1

and found that the Smad2/3 pathway is suppressed while the p38 MAPK

signaling pathway is activated in HC11FoxF1 cells.

LOX has been demonstrated to control the activity of focal adhesion kinase

(FAK) [32, 186, 189, 196]. We found that FoxF1 activates FAK in a LOX-

dependent manner, as depletion of LOX by RNAi decreased the activity of

FAK. In addition, LOX depletion activated the Smad2/3 signaling pathway

by increasing the levels of Smad2/3 and the phosphorylation of Smad2.

However, FAK inhibitor treatment showed that FAK is involved in the

activation of Smad2 but not in the regulation of total Smad2/3 levels,

suggesting that downstream of LOX there are additional factors involved in

the regulation of Smad2/3 levels. The main findings are summarized in figure

7.

Figure 7. Schematic picture of signaling pathways regulated by FoxF1.

In this study we found LOX, which is upregulated in breast carcinomas and

shown to promote metastasis, to be highly expressed in response to FoxF1

overexpression. In addition, FoxF1-induced invasiveness was shown to be

dependent on LOX activity, suggesting that induction of EMT and

invasiveness by FoxF1 may have an important role during breast tumor

progression to metastasis. Furthermore, we found FoxF1 to be involved in

TGF-β signaling pathways known to be important during breast cancer

progression. However, the possible connection between FoxF1 and TGF-β


22

and whether these factors cooperate to influence metastatic activity needs to

be further investigated.

Nuclear factor I-C2 is a powerful prognostic marker

in breast cancer (paper III).

In this study, a tissue microarray was constructed from the tissue samples of

498 patients with primary invasive breast cancer diagnosed at the Malmö

University Hospital, Malmö, Sweden, between 1988 and 1992 [197]. Of

these 498 patients the expression level of NFI-C2 could be analyzed in 462

cases, using immunohistochemistry. 217 patients stained positive and 245

stained negative for NFI-C2. The expression of NFI-C2 was positively

associated with advanced age, ERα expression and PR expression, and

negatively associated with HER2 overexpression, lymph node status, tumor

size, Ki67 expression and histological grade. This shows that NFI-C2 is

positively associated with markers for good prognosis, suggesting that tumors

with NFI-C2 expression may be less aggressive than tumors without NFI-C2

expression.

Kaplan-Meier analysis of all patients showed a significantly lower risk for

recurrence and mortality from breast cancer for NFI-C2 positive compared to

NFI-C2 negative patients, demonstrating an association between NFI-C2 and

good prognosis. However, this association was only significant for patients

with ERα-positive tumors.

Kaplan-Meier analysis of the patients that had not received any adjuvant

treatment showed that NFI-C2 was associated with increased recurrence-free

survival (RFS) and breast cancer-specific survival (BCSS), demonstrating a

prognostic value of NFI-C2. Multivariate Cox analysis including age, tumor

size, ERα, PR, Ki67, HER2, and lymph node status demonstrated that NFI-

C2 was an independent predictor of RFS. NFI-C2 remained an independent

predictor of RFS in the ERα-positive group, indicating that NFI-C2 has an

additional value in predicting breast cancer recurrence besides that of the ER

status.

NFI-C2 suppresses EMT and its expression is lost during breast cancer

progression [155], suggesting that loss of NFI-C2 may facilitate the

dissemination of carcinoma cells from the primary tumor and thereby the

generation of CTCs. It has been demonstrated that the presence of CTCs

correlates with lymph node metastases [198]. We therefore investigated the

association between NFI-C2, lymph node status and RFS in the group of

patients that had received tamoxifen treatment. The group of untreated

patients could not be analyzed due to the low number with multiple lymph

node metastases. We found that NFI-C2 was positively associated to RFS in

Gisela Nilsson

23

patients with no lymph node metastases whereas in patients with multiple

lymph node metastases, NFI-C2 was negatively associated to RFS.

Furthermore, lymph node status was only prognostic in the group of patients

with NFI-C2 positive tumors. These data suggest that the role of NFI-C2

changes during breast cancer progression.

We investigated whether tamoxifen had an effect on NFI-C2 protein levels in

two different breast cancer cell lines and found that tamoxifen reduced the

levels of NFI-C2 in 4T1 cells, known to form metastases, while it had no

effect on NFI-C2 levels in the non-invasive T47D cell line. Removing

tamoxifen from 4T1 cells restored the levels of NFI-C2.

This study supports our hypothesis that NFI-C2 is a tumor suppressor and

shows that NFI-C2 is a prognostic factor associated with good prognosis in

breast cancer. However, it also provides data suggesting that the presence of

NFI-C2 may in some instances instead be negative. NFI-C2 protein levels

were differently affected by tamoxifen which might be dependent on the

cellular phenotype. NFI-C2 has been shown to promote MET [155], which is

believed to be a critical process in metastasis formation (described in the

introduction). In patients with no lymph node metastases the presence of

NFI-C2 would have a positive effect inhibiting EMT to occur. However, in

patients with many lymph node metastases and presumably also harboring

CTCs in their blood and lymphatic system, the presence of NFI-C2 in these

circulating cancer cells might lead to MET and an increased risk for

colonization and metastases formation. Our results indicate that during

tamoxifen treatment the NFI-C2 levels are low whereas they increase when

treatment is ended. If patients who have ended a tamoxifen treatment still has

CTCs and the levels of NFI-C2 would increase, this might lead to a higher

risk for metastases formation.


24

FUTURE ASPECTS

The subject of this thesis was to investigate how different molecular factors

influence EMT. This thesis shows that c-erb2-induced EMT can proceed in

the presence of the adhesion molecule E-cadherin and that cell density-

dependent inhibition of EMT is not mediated by E-cadherin. Furthermore, it

demonstrates an involvement of NFI-C2 and FoxF1 in the regulation of

molecular factors and signaling pathways that are associated to EMT and

invasion. We identified a signaling pathway where FoxF1 upregulated LOX

in a FAK-dependent manner leading to suppression of Smad2/3 and

increased invasion. In addition, NFI-C2 was confirmed to be a prognostic

marker associated with good prognosis in breast cancer. In future work there

are several aspects that can be further investigated.

The occurrence of a mechanism that can hinder EMT is intriguing. If EMT

events are the cause of carcinoma cell dissemination and invasion,

understanding such a mechanism would give critical insights into the process

of metastases and possibly identify potential therapy targets. The effects of

cytoskeletal rearrangements and cell shape seem to be important for the EMT

process to occur and would be interesting to investigate further. Identifying

factors that induce cytoskeletal rearrangements and cell spreading (including

cell-surface targets other than E-cadherin) during EMT initiation would give

information about a possible association between cell morphology and

carcinoma progression.

To get further insight into the role of FoxF1 during carcinogenesis it would

be of importance to evaluate the effects of FoxF1 in vivo. In an ongoing

study, a transgenic mouse model for mammary carcinogenesis is used where

c-myc is specifically overexpressed in the mammary epithelium under control

of the WAP-promoter, causing tumor development after pregnancy [199]. In

this model, FoxF1 is specifically knocked-out in the epithelium by utilizing

the cre/lox system where cre is expressed under the control of the promoter of

keratin 14. This model system will give insights into the effects of FoxF1-

removal on tumor development and progression.

In addition, the 4T1 cell line can be used to study FoxF1 effect on metastatic

capability. We hypothesize that implantation of 4T1 cells overexpressing

FoxF1 into the fat pads of BALB/c mice may result in less formation of

metastases compared to control. FoxF1 overexpression would possibly result

in cancer cells with mesenchymal properties which would impede the ability

to colonize and form metastases.

Gisela Nilsson

25

The role of NFI-C2 as a prognostic marker was evaluated in a

nonrandomized trial. In order to evaluate the treatment predictive value of

NFI-C2, a randomized trial needs to be used containing a large number of

patients that has or has not received treatment.

To approach the question whether the level of NFI-C2 protein affects the

tumor cells' capacity to metastasize, the 4T1 cell line can be utilized. 4T1

cells where NFI-C2 has been knocked-down and control cells can be

implanted into the fat pads of BALB/c mice and their metastatic capability

compared. If NFI-C2 promotes MET then the NFI-C2 knock-down cells will

be more mesenchymal, and as a consequence of that, less capable to colonize

and form metastases.

To elucidate the dual effects of NFI-C2 positivity depending on tumor

progression stage, one can overexpress NF1-C2 in 4T1 cells. Implantation of

these cells in the mammary fat pads of BALB/c mice might give rise to

tumors with less metastatic capability compared to control. On the other

hand, if NFI-C2 overexpressing cells instead were injected into the tail,

resulting in cells entering the bloodstream, this might instead increase the

metastatic capability. We hypothesize that NFI-C2 overexpression would

generate cancer cells with epithelial properties enabling attachment at distant

sites and metastasis formation.


26

ACKNOWLEDGEMENTS

I would like to thank all the people who in different ways have contributed to

this thesis. In particular I would like to thank:

Dan Baeckström, my supervisor, for taking me on as a PhD student in the

organized DB-lab, for all your support, for always taking time for questions

and for your positive thinking.

The former members of the DB-lab: Shahram for being a good friend and

for all the help in the beginning, Karin for nice company and for always

being positive and Noreen for nice collaboration.

Marie Kannius Janson, my co-supervisor, and Jeanette Nilsson, for letting

me continue my PhD studies in your lab and for your support and

encouragement. It has been a very educating and fun period for me.

Elin for teaching me immunohistochemistry and mouse models and for being

a good friend.

Levent Akyürek and Staffan Johansson for your help during the period

when you were my co-supervisors.

I would like to thank all my former co-workers at medkem, especially:

Gunnar Hanson for your support and for letting me use your equipment, and

all former and present members of the MUCIN-lab.

Isabella, Monica and Ka-Wei for technical advice and nice company during

my time at medkem.

Erika, Daniel, Nicola, Anna and Muna for nice fika-times.

I would also like to thank my co-workers at CMB, especially:

Peter Carlsson for valuable comments and for sharing your Fox-expertise.

Ali and Azadeh for sharing your knowledge and for contributing to the

friendly environment at CMB.

All the people in Per Sunnerhagens lab. Johanna for stimulating

discussions and for making me such beautiful pottery.

Marc Pilon, Julie Grantham and everyone in your labs for contributing to

the stimulating and enjoyable environment at CMB.

Catarina for being such a kind and fun person.

Valida for keeping the lab tidy.

Cecilia, it was nice to have you around all these years.

Gisela Nilsson

27

My mother and father for always being there for me and my family, without

you this would not have been possible. Veronica and Bobby for being the

best siblings. Also, thanks Veronica for all the help during my last period of

writing.

Maria and Monica, my beautiful friends, for your interest in my work and

for sharing fun times outside work.

Daniel for your love and support. Polly and Elliot, I love every day I get to

spend with you!


28

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Molecular factors influencing epithelial … factors influencing epithelial-mesenchymal transition in breast cancer Gisela Nilsson Department of Medical Biochemistry and Cell Biology,